Cell Biology

Lipids can play diverse roles in metabolism, signaling, transport across membranes, regulating body temperature, and inflammation. Some viruses have evolved to exploit lipids in human cells to promote viral entry, fusion, replication, assembly, and energy production through fatty acid beta-oxidation. Hence, studying the virus–lipid interactions provides an opportunity to understand the biological processes involved in the viral life cycle, which can facilitate the development of antivirals. Due to the diversity and complexity of lipids, the assessment of lipid utilization in infected host cells can be challenging. However, the development of mass spectrometry, bioenergetics profiling, and bioinformatics has significantly advanced our knowledge on the study of lipidomics. Herein, we describe the detailed methods for lipid extraction, mass spectrometry, and assessment of fatty acid oxidation on cellular bioenergetics, as well as the bioinformatics approaches for detailed lipid analysis and utilization in host cells. These methods were employed for the investigation of lipid alterations in TMEM41B- and VMP1-deficient cells, where we previously found global dysregulations of the lipidome in these cells. Furthermore, we developed a web app to plot clustermaps or heatmaps for mass spectrometry data that is open source and can be hosted locally or at https://kuanrongchan-lipid-metabolite-analysis-app-k4im47.streamlit.app/. This protocol provides an efficient step-by-step methodology to assess lipid composition and usage in host cells.

Graphical overview

The ability to stain lipid stores in vivo allows for the facile assessment of metabolic status in individuals of a population following genetic and environmental manipulation or pharmacological treatment. In the animal model Caenorhabditis elegans, lipids are stored in and mobilized from intracellular lipid droplets in the intestinal and hypodermal tissues. The abundance, size, and distribution of these lipids can be readily assessed by two staining methods for neutral lipids: Oil Red O (ORO) and Nile Red (NR). ORO and NR can be used to quantitatively measure lipid droplet abundance, while ORO can also define tissue distribution and lipid droplet size. C. elegans are a useful animal model in studying pathways relating to aging, fat storage, and metabolism, as their transparent nature allows for easy microscopic assessment of lipid droplets. This is done by fixation and permeabilization, staining with NR or ORO, image capture on a microscope, and computational identification and quantification of lipid droplets in individuals within a cohort. To ensure reproducibility in lipid measurements, we provide a detailed protocol to measure intracellular lipid dynamics in C. elegans.

Graphic abstract:

Flow chart depicting the preparation of C. elegans for fat staining protocols.

The endothelial cells from the microvasculature are key drivers and targets of inflammatory and thrombotic processes in microvascular diseases. The study of bioactive lipids in inflammatory processes has been largely based on two-dimensional endothelial cell cultures. Three-dimensional microvessels-on-a-chip provides an opportunity to monitor the inflammatory phenotype of human microvessels in a more physiological-relevant environment. This protocol describes the culture of endothelial cells as three-dimensional microvessels in the OrganoPlate. The microvessels are treated with tumor necrosis factor alpha to induce inflammation. The collection of samples from the microvessels is optimized for measuring bioactive lipids with liquid chromatography-mass spectrometry, providing a more informative metabolic readout as compared with functional assays.

Sphingolipids are major structural components of endomembranes and have also been described as an intracellular second messenger involved in various biological functions in all eukaryotes and a few prokaryotes. Ceramides (Cer), the central molecules of sphingolipids, have been depicted in cell growth arrest, cell differentiation, and apoptosis. With the development of lipidomics, the identification of ceramides has been analyzed in many species, mostly in model insects. However, there is still a lack of research in non-model organisms. Here we describe a relatively simple and sensitive method for the extraction, identification, and quantification of ceramides in Hemiptera Insects (brown planthooper), followed by Ultra-Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). C18 is used as the separation column for quantitative detection and analysis on the triple quadruple liquid mass spectrometer. In this protocol, the standard curve method is adopted to confirm the more accurate quantification of ceramides based on the optional detection conditions.

Macrophages are highly plastic immune cells that are capable of adopting a wide array of functional phenotypes in response to environmental stimuli. The changes in macrophage function are often supported and regulated by changes in cellular metabolism. Capturing a comprehensive picture of metabolism is vital for understanding the role of metabolic rewiring in the immune response. Here we present a method for systematically quantifying the abundance of metabolites and lipids in primary murine bone marrow derived macrophages (BMDMs). This method simultaneously extracts polar metabolites and lipids from BMDMs using a rapid two-phase extraction procedure. The polar metabolite fraction and lipid fraction are subsequently analyzed by separate liquid chromatography-mass spectrometry (LC-MS) methods for optimized coverage and quantification. This allows for a comprehensive characterization of cellular metabolism that can be used to understand the impact of a variety of environmental stimuli on macrophage metabolism and function.

Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and animal cells and comprehensively analyse their lipid composition.

Lipid droplets (LDs) are central organelles in maintaining lipid homeostasis. Defective LD growth often results in the development of metabolic disorders. LD fusion and growth mediated by cell death–inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins are crucial for various biological processes including unilocular LD formation in the adipocytes, lipid storage in the liver, milk lipid secretion in the mammary epithelia cells, and lipid secretion in the skin sebocytes. Previous methodology by Gong et al. (2011) first reported a lipid-exchange rate assay to evaluate the fusion ability of each LD pair in the cells mediated by CIDE family proteins and their regulators, but photobleaching issue remains a problem and a detailed procedure was not provided. Here, we provide an improved and detailed protocol for the lipid-exchange rate measurement. The three key steps for this assay are cell preparation, image acquisition, and data analysis. The images of the fluorescence recovery are acquired after photobleaching followed by the measurement of the intensity changes in the LD pair. The difference in fluorescent intensity is used to obtain the lipid exchange rate between the LDs. The accuracy and repetitiveness of the calculated exchange rates are assured with three-cycle of photobleaching process and the linear criteria in data fitting. With this quantitative assay, we are able to identify the functional roles of the key proteins and the effects of their mutants on LD fusion.

It has been well-established that malondialdehyde (MDA), which is generated during the process of lipid peroxidation, is a commonly known biomarker for oxidative stress. Therefore, the serum levels of MDA are detected by using the lipid peroxidation assay with commercially available kit to determine the induction of oxidative stress in rat models.

Almost all functions of cells or organs rely on the activities of cellular enzymes. Indeed, the in-vivo activities that directly represent the cellular effects of enzymes in live organs are critical importance to appreciate the roles enzymes play in modulating physiological or pathological processes, although assessments of such in-vivo enzyme activity are more difficult than typical test-tube assays. Recently, we, for the first time, developed a direct and easy-handling method for HPLC analyzing the in-vivo activity of glucosylceramide synthase (GCS). GCS that converts ceramide into glucosylceramide is a limiting-enzyme in the syntheses of glycosphingolipids and is one cause of cancer drug resistance. In our method developed, rubusoside nanomicelles delivers fluorescence N-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphingosine (NBD C₆-ceramide) into mice, tissues uptake the cell-permeable substrate, and GCS converts it into NBD C₆-glucosylceramide in all organs simultaneously. Further, HPLC analyzes the extracted NBD C₆-glucosylceramide to assess alterations of the in-vivo GCS activities in tissues. This method can be broadly used to assess the in-vivo GCS activities in any kind of animal models to appreciate either the role GCS plays in diseases or the therapeutic efficacies of GCS inhibitors.

Studying lipid metabolism in cultured cells is complicated by the fact that cells are typically cultured in the presence of animal serum, which contains a wide, variable, and undefined variety of lipid species. Lipid metabolism can impact cell physiology, signaling, and proliferation, and the ability to culture cells in the absence of exogenous lipids can reveal the importance of lipid biosynthesis pathways and facilitate the generation of media with defined lipid species. We have adapted a protocol to remove lipids from serum without eliminating its ability to support the proliferation of cells in culture. This method requires di-isopropyl ether and butanol and can be used to generate small batches of lipid-stripped serum in four days. The resulting serum supports proliferation of many cell lines in culture and can be used to compare the metabolism of cells in lipid replete and depleted conditions.